Ischemic heart disease is one of the most common causes of death in the United States and is responsible for a tremendous healthcare burden. Cardioprotective interventions hold great promise for lessening this burden, but few experimental discoveries have translated successfully into effective therapeutics. This is likely due to a lack of knowledge with regard to the mechanistic details of cardioprotection. Nitric oxide, either produced endogenously or administered exogenously, has been shown to be an important component of cardioprotection. Our recent studies have demonstrated an association between increased levels of nitric oxide-derived protein S-nitrosylation and cardioprotection. S-nitrosylation is a reversible, thiol-based modification that is produced from the covalent attachment of a nitric oxide moiety to the free thiol group of a cysteine residue. The nitric oxide synthase isoforms represent the major source of endogenous nitric oxide production in the cardiac myocyte. S-nitrosylation is thought to provide protective effects by modulating the activity and/or function of target proteins, and by blocking the damaging effects of irreversible cysteine oxidation. Our recent data are consistent with an overall protective role for S-nitrosylation, but the molecular mechanism(s), the relative importance of specific protein targets, and the pathophysiological significance of S- nitrosylation is unknown. Thus, the goal of this proposal is to define the specificity and mechanistic consequences of protein S-nitrosylation in the myocardium by investigating the physiologic and pathologic aspects of S-nitrosylation. To establish the mechanistic details of S-nitrosylation, a novel method to determine the percentage of a given protein that is modified by S-nitrosylation (i.e., S-nitrosylation occupancy) is being developed and this will be used to examine compartmentalized S-nitrosylation signaling. Cysteine mutagenesis will also be used to examine the effects of S-nitrosylation on protein function. We previously identified many S- nitrosylation sites in cardioprotection, thus allowing us to focus on specific cysteine residues that are altered in situ. The specific aims of this proposal are as follows: 1) determine if S-nitrosylation occupancy varies between different cellular compartments of the cardiomyocyte and evaluate how this affects signaling, 2) determine if S- nitrosylation changes protein-protein interaction, alters protein localization, and promotes protein trans-S- nitrosylation, and 3) determine the specific role of S-nitrosylation in the regulation of myocardial ion channel activity. The results of this proposal will define the rol of S-nitrosylation in the heart and advance the field by establishing a mechanistic role for S-nitrosylation, thus lending critical insight into potential therapeutic targets.